Deferration using anguibactin siderophore

ABSTRACT

Methods for removing ferric iron from aqueous liquids and for performing deferration therapy are disclosed, involving the use of a novel siderophore, termed anguibactin. Anguibactin is isolated from a marine pathogen, Vibrio anguillarum, containing the pMJ1 plasmid. Anguibactin inhibits iron uptake by living cells, wrests iron from vertebrate tissues, removes iron from other siderophores and ferric hydroxide, and removes ferric iron from aqueous solutions, including cell-culture media. For deferration therapy, anguibactin from which bound iron has been removed is administered by dissolving in water or other liquid aqueous pharmaceutical carrier at a dosage typical for other siderophores. Anguibactin is preferably administered intramuscularly or subcutaneously, but can be given intravenously. Oral administration is also possible, particularly if the siderophore is encapsulated in a form allowing it to pass intact through the acidic environment of the stomach but become available for absorption in the intestine. This siderophore has the advantages of low molecular weight, extremely high affinity for ferric iron, and non-use by any known human pathogen. The structure is amenable to immobilization on a solid substrate.

This invention was funded in part by Public Health Service Grant No.AI19018 from the National Institute of Allergy and Infectious Diseases,National Institutes of Health. The government has rights in thisinvention.

FIELD OF THE INVENTION

This invention relates to removing ferric iron from aqueous liquidsusing novel compositions of matter. In particular, it relates to using anovel siderophore for deferration therapy and other relatedapplications.

BACKGROUND OF THE INVENTION

It has been known for years that many bacteria require iron for growth.At least some types of bacteria and fungi obtain the iron they need byproducing special compounds termed "siderophores" (Greek for "ironbearers") which are relatively low molecular-weight (less than about1000 daltons) iron-binding ("iron chelating") compounds. Generally,siderophores are ferric-specific ligands, the natural purpose of whichis to supply iron to the microorganism cells. Each of the severalspecies of siderophores is a key component in the iron high-affinitysystem of the respective microorganism that includes specificmembrane-associated receptors.

Representative siderophores include phenolate compounds such as"agrobactin" from Agrobacterium tumefaciens and "pseudobactin" fromPseudomonas, and hydroxamates such as "schizokinin" from Bacillusmegaterium and ferrioxamines from Actinomyces. Siderophores produced byfungi include hydroxamates such as ferrichromes from Penicilliumspecies, rhodotorulic acids from Rhodoturula, and other hydroxamatesfrom certain Ectomycorrhiza species. See Neilands, Ann. Rev. Biochem.50:715-731 (1981).

Host animals such as mammals and fish produce iron-binding proteins,including ferritin, transferrin, and hemosiderin, which tightlysequester ionic iron in the body. As a result, unbound or "free" iron(as ferrous or ferric ions) is present only at very low concentrationsin a healthy host's plasma and other body fluids.

Siderophore production enables invading bacteria to successfully competewith the host's iron-binding proteins for iron in the host's body thatwould otherwise be unavailable to the bacteria. Without an ability towrest bound iron from the host, bacteria would be unable to proliferatesufficiently to cause disease. Hence, production of siderophores is akey to bacterial pathogenicity.

A number of diseases in humans are demonstrative of the toxicity of freeiron in the body. In general, the term "hypersiderosis" represents anyof several disease conditions in which the normal iron-carrying capacityof a person's blood and tissue proteins is exceeded and pathologicaleffects due to iron overload are manifest. In such conditions, theexcess iron can become deposited in various tissues, such as themyocardium and liver.

Acute iron intoxication usually results from accidental over-ingestionof iron supplements, particularly by young children. Industrialaccidents can also result in acute iron intoxication.

Chronic iron overload encompasses a variety of diseases where ironaccumulates in the body due to various causes. For example, intestinalcontrol of iron absorption may be ineffective so that inappropriateamounts of dietary iron are allowed to enter the body (e.g., idiopathichemochromatosis and anemias with ineffective erythropoiesis). In suchcases, iron overload develops even when a normal diet is consumed.Hemochromatosis can also occur in alcoholics with cirrhosis. Long-termexposure to a diet containing excessive iron can lead to iron overloadin otherwise normal subjects (dietary iron overload). In addition, largeamounts of parenteral preparations of iron inappropriately prescribed,or repeated blood transfusions for refractory anemias, may result in theaccumulation of excess iron in the body (transfusional siderosis). Theiron liberated from the transfused cells cannot be excreted and itaccumulates in the cells of the reticuloendothelial system and incardiac muscle, kidneys, thyroid gland and adrenal gland. Changes iniron distribution from the primary reticuloendothelial iron toparenchymal iron overload are ascribed to the high saturation oftransferrin, which provides favorable conditions for uptake of iron byparenchymal cells. Free transferrin thus protects the tissues fromsiderosis.

An example of a hereditary disease characterized by chronic ironoverload is Cooley's anemia (thallasemia major), where congestive heartfailure often precedes rapid deterioration and death of the untreatedpatient almost always in early infancy.

Electrocardiogram abnormalities are the most frequent manifestations ofthe cardiomyopathy of hemochromatosis. In descending order of frequency,these are T-wave flattening and inversion, low-voltage tracings,arrhythmias both superventricular (notable auricular fibrillation)and/or ventricular (premature ventricular contractions which may precedeventricular tachycardia or ventricular fibrillation). Congestive heartfailure is rarer, but may be fatal, especially in young subjects.Postmortem examination of the heart shows fibrosis and hemosiderindeposits which are greater in the ventricles than in the atria, greateron the left side than the right side, and greater in the epicentrum thanin the endocardium. Iron chelation therapy offers the possibility ofalleviating this harmful and potentially lethal accumulation of iron incardiac tissue.

Virtually the only iron chelator or siderophore currently inpharmacological use is deferoxamine (DESFERAL from CIBA Pharmaceuticals;U.S. Pat. Nos. 3,118,823 and 3,153,621). Deferoxamine was originallyisolated from Streptomyces pilosus. This drug chelates iron by forming astable complex with an iron atom. The complex prevents the iron fromentering into further chemical reactions. The drug has a high affinityfor ferric iron (K_(a) =10³¹) coupled with a very low affinity forcalcium (K_(a) =10²). Deferoxamine wrests iron from ferritin andhemosiderin but not readily from transferrin and substantially not atall from cytochromes and hemoglobin. Theoretically, deferoxamine iscapable of binding about 8.5 parts by weight of ferric iron. Themolecular weight of deferoxamine is 657 g/mol.

Deferoxamine has improved the prognosis for iron-overload patients.However, this drug has certain drawbacks. First, the drug is prone toacid hydrolysis and poorly absorbed after oral administration, requiringparenteral administration, which is particularly inconvenient forlong-term therapy. Second, it becomes effective when the body's ironload is at least about ten times normal, which is a level at whichiron-binding proteins in the body are saturated and toxic free iron iscirculating in the body. Third, it is expensive to produce. At thepresent time, the cost of sufficient deferoxamine for a year's treatmentof chronic iron overload is several thousand dollars. Fourth,deferoxamine is toxic and can cause a number of reactions, includingallergic reactions: pruritis, wheals, rash, and anaphylaxis; anddysuria, gastrointestinal symptoms, diarrhea, fever, leg cramps,hypotension, and tachycardia. Intravenous LD₅₀ values are 287 mg/kg inmice; 329 mg/kg in rats.

Another major disadvantage of deferoxamine is its use by certainmicroorganisms to enhance their pathogenicity in humans. For example,iron overload increases the susceptibility of patients to Yersiniaenterocolitica infections. In some cases, treatment with deferoxaminehas enhanced this susceptibility, resulting in generalized infections byproviding this bacterium with a siderophore otherwise missing. In suchcases, deferoxamine treatment must be discontinued until the infectionis resolved.

Examples of siderophores that have not achieved the pharmaceuticalpopularity of deferoxamine include catechol derivatives as disclosed inU.S. Pat. Nos. 4,530,963 and 4,585,559 to DeVoe et al., andhydroxypyridone derivatives as disclosed in Hider et al., U.S. Pat. No.4,666,927.

Hence, there is a need for a new pharmacological method for reducing theconcentration of ferric iron in the body, for treating iron overload andrelated diseases, particularly by using a new siderophore that isrelatively non-toxic, producible at low cost, and not utilizable by anyknown human pathogens.

SUMMARY OF THE INVENTION

The above-stated need is addressed by the pharmacological use ofanguibactin, a siderophore having an unusual chemical structure producedby a marine pathogen, Vibrio anguillarum. Anguibactin is producible inlarge quantities at low cost as a result of the cloning of a plasmidcomprising a normal iron-uptake region of V. anguillarum. Anguibactin isresistant to acid hydrolysis at an acid pH as low as 3 and is soluble inwater and methanol.

Anguibactin inhibits iron uptake by living cells, as determined inexperiments using diploid human fibroblasts and rat heart cells.Anguibactin is also able to wrest iron from the tissues of a fish hostsufficient for growth and virulence of V. anquillarum and to remove ironfrom other siderophores such as aerobactin, and transferrin, as well asfrom ferric hydroxide. Anguibactin also apparently has very lowtoxicity, has a molecular weight about half that of deferoxamine andappears to chelate ferric iron more strongly than deferoxamine.Anguibactin is also capable of removing ferric iron from aqueoussolutions, including cell-culture medium.

For deferration therapy, the dose regimen of anguibactin would typicallystart out high and be reduced as therapy progresses. Initial dosageswould be within the range of 0.1 to 5 g, preferably about 0.5 to 2.5 g,which is the customary range of siderophore dosages for human use,depending in part upon the age, weight, and degree of iron intoxicationof the patient. For example, a 200-pound person would initially receivea dose of about 11 mg/kg, followed by doses every 4-6 hours thereafterof about 5.5 mg/kg. Veterinary dosages would be based on g/kg weightratios similar to those for humans. Prior to pharmacogical use, boundferric iron should be removed from the siderophore to ensure maximalchelating capacity of the dose, which can be performed by subjectingferrated anguibactin to a reducing agent serving to reduce the chelatedferric ion to a ferrous ion.

To protect orally administered anguibactin against acid hydrolysis in asubject's stomach, anguibactin can be rendered resistant to acid attackby encapsulating in microcapsules able to pass intact through thestomach and yet disintegrate upon reaching the neutral or slightlyalkaline environment of the subject's intestine where absorption of thedrug into the subject's body occurs.

Anguibactin has a chemical structure amenable to immobilizing theanguibactin molecules on a solid substrate so as to employ thesiderophore in columns and the like for deferration of liquids includingbody fluids.

It is accordingly one object of the present invention to provide animproved method for reducing the concentration of ferric iron in anaqueous medium contacting a population of living eukaryotic cells.

Another object of the present invention is to provide a method forinhibiting the uptake of ferric iron by a population of livingeukaryotic cells.

Another object of the present invention is to provide an improved methodfor reducing the concentration of ferric iron in the body of avertebrate animal subject.

Another object of the present invention is to provide an improved methodfor removing ferric iron from a liquid.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a genetic map of the iron-uptake region of the pJM1 plasmidpresent in virulent strains of Vibrio anguillarum.

FIG. 2 is a schematic depiction of the structural formula ofanguibactin.

FIG. 3 is a plot showing that anguibactin inhibits uptake, from a liquidmedium, of iron by cultured human diploid fibroblast cells.

DETAILED DESCRIPTION

Virulent strains of the fish pathogen Vibrio anguillarum are able toproduce a distinctive water-soluble siderophore termed "anguibactin".Actis et al., J. Bacteriol. 167:57 (1986); Crosa et al., in Helinski etal. (eds.), Plasmids in Bacteria, pp. 759-774, Plenum, N.Y. (1985).Anguibactin is very efficient in removing iron from aqueous solution aswell as from transferrin and from other iron-binding proteins in thefish host. The discovery of this siderophore resulted from theobservation that the main difference between virulent and non-virulentstrains was the ability of the former to grow invasively at lowconcentrations of iron.

Virulent strains of Vibrio anguillarum, which cause a terminalhemorrhagic septicemia in fish, possess a 65 kbp (65000 base pairs)plasmid named pMJ1 absent from non-virulent strains. Mutational studiesestablished that pMJ1 carries the genes for an inducible high-efficiencyiron uptake system which facilitates virulence. This iron-uptake systemis induced in pMJ1-containing strains during growth under iron-limitedconditions. Induction of the system results in the energy-dependentuptake of iron by the V. anguillarum cells.

Cloning of pMJ1 Plasmid Comprising Anguibactin Genetic Elements

The plasmid pMJ1 has now been cloned, the genetic map of the iron-uptakeregion of which is shown in FIG. 1. The genetic units I-VI comprisingthe iron-uptake region, occupying about 25 kilobase pairs (kbp) of pMJ1,were defined by transposition mutagenesis as reviewed in Crosa,Microbiol. Rev., Dec. 1989, pp. 517-530. The iron-uptake system includesan 86-kilodalton (kDa) outer membrane protein pOM2, the presence ofwhich is associated with the acceptance and transport of iron into thecell cytosol. The pOM2 protein is missing from strains unable to grow inmedia in which iron is complexed by nonassimilable iron chelators, evenwhen the strains are supplied with additional anguibactin purified fromwild-type cells.

As seen in FIG. 1, anguibactin synthesis is encoded by genetic units I,IV, V, and VI. The ORF5 region (genetic unit III) encodes a regulatoryprotein which functions in a complex scheme for expression of theiron-uptake system, as detailed in Crosa, Microbiol. Rev., December1989, pp. 517-530.

Cloning of pJM1 has permitted anguibactin to be produced in largequantities at relatively low cost.

Culture of V. anguillarum

Cells of Vibrio anguillarum 775 were grown for 48 hours in M9 minimalmedium, Crosa, Nature (London) 283:566-568 (1980), containing 100 μM ofthe nonassimilable iron chelator nitriloacetic acid. To remove traces ofcontaminating metals, medium salts were passed through a Chelex 100column prior to use. Bacteria were separated from the medium bycentrifugation at 7,000×g. Supernatants were stored at -20° C.

Purification of Anguibactin

Anguibactin was isolated from V. anguillarum 775 supernatants byadsorption onto XAD-7 macroreticular resin (Rohm and Haas). This resinwas subjected to sequential Soxhlet extractions with methanol,acetonitrile, and diethylether to remove residual organic impuritiesprior to being packed into a column in water. The pH of culturesupernatants was adjusted to neutrality to avoid coadsorption oflow-molecular-weight organic acids. A 10-liter volume of supernatant wasthen applied to the packed XAD-7 column (5 by 10 cm) at a rate of 10 bedvolumes per hour. The column was rinsed with two void volumes ofdeionized water, followed by a step gradient of one void volume each of1:2 and 2:1 (v/v) methanol:water mixtures. Adsorbed material in thecolumn was eluted with pure methanol. The peak column fractionscontaining anguibactin were evaporated at reduced pressure, and theresidue was dissolved in 3 mL of methanol. This material was thenapplied to a column (1.5 by 80 cm) of Sephadex LH-20 (Pharmacia) andeluted with methanol at 0.6 mL/min. Peak fractions from the LH-20 columnwere reduced to dryness by rotary evaporation followed by exposure tohigh vacuum for one hour and stored under nitrogen at -20° C.

Biologically active anguibactin was assayed in supernatants and columnfractions from the size of a growth halo around a 7-mm diameter sterilefilter disk on an agarose plate containing M9 minimal medium plus 15 μMEDDA [ethylenediamine-di(o-hydroxyphenylacetic acid)] that had beenseeded with 0.1 mL of an overnight culture of 10⁸ cells of V.anguillarum strain 775::Tn1-5. This mutant strain contains the mutantplasmid pJHC-91 (explained infra), which is receptor-proficient butsiderophore-deficient. A control for siderophore specificity was thelack of a growth halo with strain H775-3 which is missing the pJM1plasmid (explained infra) and is therefore deficient in receptor as wellas siderophore activity. Typically, 5 μL of the test solution wasspotted on the filter disk and allowed to dry before the disk wasapplied to the agarose plate. The diameter of the halo was measuredafter growth for 24 to 48 hours at 25° C., and that value was correctedby subtracting the 7-mm contribution from the filter disk.

Anguibactin concentrations in column fractions were quantified bymeasuring the strong UV absorbance of the siderophore in acidic ironperchlorate. Methanolic solutions were evaporated to dryness under astream of N₂ and dissolved in 0.5 mL of deionized water, and then 0.5 mLof 5 mM FeCl₃ in 0.14M HClO₄ was added. The absorbance of the assaysolution at 307 nm (the adsorption maximum of anguibactin) was measuredagainst a blank reference containing water:FeCl₃ -HClO₄ (1:1).

Structure and Properties of Anquibactin

The structure of anguibactin was determined as outlined in Jalal et al.,J. Am. Chem. Soc. 111:292 (1989), and shown schematically in FIG. 2.Anguibactin has a molecular weight of 348 g/mol, which is about half themolecular weight of deferoxamine (657 g/mol). Anguibactin can beregarded as a form of catechol rather than a monophenol. It has a uniquestructure which bears some resemblance to pyochelin. See Llinas et al.,Biochemistry 12:3836 (1973). Anguibactin has been identified asω-N-hydroxy-ω-[[2'-(2'',3''-dihydroxyphenyl)thiazolin-4'-yl]-carboxy]histamineby crystal X-ray diffraction studies of its anhydro derivative, protonand ¹³ C nuclear magnetic resonance spectroscopy of its deferri andGa(III) complex, fast-atom bombardment (FAB) mass spectrometry, andchemical degradation. As can be seen, the molecule contains catecholateand hydroxamate structures. Single-crystal structure determination ofthe Ga(III) complex (used instead of iron) of racemized anguibactinshowed a 1:1 metal-to-ligand stoichiometry in which the O-hydroxy group,the nitrogen of the thiazolin ring, the hydroxamate (N-O group), and thedeprotonated nitrogen of the imidazole ring coordinate the metal ion.

Each molecule of anguibactin chelates one ferric (Fe³⁺ or Fe(III)) ion.Anguibactin also binds Ga(III). It is expected that anguibactin, similarto other siderophores such as ferrichrome and enterobactin that bindFe(III) and Ga(III), can also bind Ai(III), but tests to determine thishave not yet been performed. Anguibactin does not bind any metal otherthan iron known to be essential for bacterial metabolism. Also,anguibactin has only a very weak affinity for ferrous (Fe²⁺) and Ca²⁺ions.

As expected, the ability of anguibactin to bind F³⁺ is dependent on pH.However, excellent binding can be achieved even in mildly alkalineconditions. The affinity of anguibactin for ferric ions is extremelyhigh, as evidenced by the ability of anguibactin to remove ferric ionfrom ferric hydroxide which is extremely insoluble in aqueous solutionsat pH between 7 and 8 (K_(s) <10⁻³⁸ M). In competitive binding assays,anguibactin is able to remove Fe³⁺ from other siderophores such asaerobactin, which has a deferrisiderophore formation constant (logK_(f)) of about 22.9, and from transferrin, which has a log K_(f) valuewithin the range of about 32 to about 36. Since deferoxamine has adeferrisiderophore formation constant of about 30.6, these data indicatethat anguibactin is a more powerful iron chelator than the widely useddeferoxamine.

The small size of anguibactin, relative to other siderophores such asdeferoxamine, is believed to be a factor contributing to its strongability to wrest Fe(III) from iron-transport and iron-storage proteins.Metal-protein bonds act over small distances and a bound metal ion isoften buried in a cleft or the like in the protein molecule. Anysuccessful "competitor" chelator must be able to penetrate to a positionclosely adjacent the protein-metal bond so as to disrupt it and permitthe metal ion to pass over to the chelator. The smaller the chelator,the generally better its "penetrating" ability.

Anguibactin is freely soluble in water and methanol. Although acidhydrolyzable under certain acid conditions, the anguibactin moleculeappears to remain intact at a pH as low as 3. Hydrochloric acid at aconcentration of 6N will cleave anguibactin to 2,3-dihydroxybenzoicacid, dehydrocystine, and histamine.

Bacterial Production of Diffusible Anquibactin

A mutant strain of Vibrio anguillarum termed 775:Tn1-5 was producedwhich contained the pJM1-derivative plasmid pJHC-91 in which atransposon element Tn1 was inserted into genetic unit I. This mutantstrain can grow in vitro in iron-limited media only if the supernatantfrom strains containing the wild-type pJM1 plasmid, and thus plenty ofanguibactin, is supplied. Therefore, strains harboring the pJHC-91plasmid must be able to transport and incorporate iron from anguibactinbut are not able to produce this anguibactin itself. Strains containingthe pJHC-91 plasmid are able to synthesize the pOM2 outer membraneprotein.

Another mutant of V. anguillarum, 775:Tn1-6, harboring a variant ofplasmid pJM1 termed pJHC9-8, lacks both the ability to synthesizeanguibactin and the ability to use the siderophore when it is suppliedfrom external sources. Plasmid pJHC9-8 is a derivative of pJM1 thatresulted from Tn1 insertion and deletion of most of the iron-uptakeregion. Strains harboring pJHC9-8 not only lack the ability to produceanguibactin but also do not synthesize the pOM2 protein.

Experimental infections of salmonid fishes with mixtures consisting of awild-type virulent strain of V. anguillarum and thesiderophore-deficient, receptor-deficient mutant strain 775::Tn1-5resulted in recovery of both the wild-type strain and the mutant strain,whereas infections with mixtures consisting of the wild-type virulentstrain and the siderophore-deficient, receptor-deficient mutant775::Tn1-6 resulted in recovery of only the wild-type strain. Theseresults demonstrated that anguibactin is produced in vivo in adiffusible form by V. anguillarum.

The level of siderophore in the blood and kidneys of fish infected withthe wild-type strain was sufficient to provide iron for considerablegrowth of the avirulent strain lacking the ability to produce thesiderophore but possessing the transport functions. These resultsindicate that anguibactin is released from cells infected with V.anguillarum, encounters ferric iron bound to iron-transport proteins inthe host's body, and forms a ferri-anguibactin complex by strippingFe(III) from said proteins. The complex is then utilized by bothwild-type bacteria and uptake-proficient bacteria after contact with areceptor protein to which the ferri-anguibactin complex binds.

Inhibition by Anguibactin of Iron Uptake by Cells

An experiment was conducted to characterize the chelation properties ofanguibactin by examining its effect on iron uptake by mammalian cells inculture.

Normal human diploid fibroblast cells were mixed with ⁵⁹ Fe-containingcell-growth medium. Samples of the dosed cells were then incubated forincreasing lengths of time. After each time period, the correspondingsamples were rinsed to remove any free radioiron. The rinsed cells weretreated with trypsin and the lysates precipitated with trichloroaceticacid. Radioactive counting of the precipitated lysates was performedusing a Beckman counter. Addition of anguibactin and subsequent assayfor radioiron in otherwise identical parallel samples yielded a measureof the capacity of the siderophore present in the cell-growth medium tointerrupt the uptake of radioiron by the cultured cells. Results areshown in FIG. 3, which clearly shows that anguibactin interrupts theflow of iron into human cells.

A similar experiment performed using rat heart cells not yet"immortalized" for sustained growth under cell-culture conditionsyielded substantially the same results (data not shown).

These results, considered in combination with the results of experimentsdemonstrating production of diffusible anguibactin able to wrestsufficient iron from tissues of a fish host sufficient for bacterialgrowth and virulence (supra), indicate that anguibactin is effective ininhibiting the iron uptake of cells of animals as diverse as humans,rats, and fish. As a result, anguibactin would be effective as asiderophore administered to a subject animal for the purpose of removingexcess iron from the subject's body.

Toxicity of Anguibactin

Preliminary studies indicate that anguibactin toxicity is very low.Injection of 400 μl anguibactin into fish and 400 μl into rabbits showedno discernable adverse effect on the subjects. These results may beexplained in part by the low affinity of anguibactin for Ca²⁺. As isknown in the art, calcium chelators such as EDTA are relatively toxic inpart because they can cause a depletion of Ca²⁺ in body fluids, whereCa²⁺ is vital for many normal metabolic processes.

Evaluating Anguibactin for Use in Deferration Therapy

Since anguibactin is highly soluble in water, apparently has very lowtoxicity, has a molecular weight about half that of deferoxamine,impairs uptake of radioiron by living cells, and appears to chelateferric iron even more strongly than deferoxamine, it is expected thatanguibactin would be an effective siderophore for deferration therapy.

Similar to deferoxamine and other "hard"-base siderophores, anguibactinshows little affinity for "soft" acid cations such as Fe²⁺. As a result,ferric iron can be released from the siderophore via a reduction step,wherein the chelated ferric iron is reduced to ferrous iron (Fe²⁺) whichis then released from the siderophore. Such release of iron prepares themolecules for use as an iron chelator drug. For example, sodium andpotassium dithionites (hyposulfites), and sodium and potassiumascorbates are known in the art as suitable reducing agents,particularly for hydroxamates and phenolates or catecholates,respectively. Other candidate reducing agents are hydroquinone andhydroxylamine.

Anguibactin is administered in vivo to a subject as an aqueous solution,preferably by intramuscular or subcutaneous injection, in a regimensimilar to that used for deferoxamine. For intramuscular or subcutaneousadministration, anguibactin is prepared by dissolving the purifiedcompound in pyrogen-free sterile water or isotonic saline at aconcentration of about 250 mg/mL. If administered intravenously, itshould be added to a standard I.V. solution such as isotonic saline oraqueous glucose solution. For deferration therapy, the dose regimenwould typically start out relatively high and then be reduced as therapyprogresses. Siderophore dosages for human use typically range from 0.1to 5 g, preferably 0.5 to 2.5 g, depending in part upon the age, weight,and degree of iron intoxication of the patient. With deferoxamine, 1.0 gis usually administered initially, followed by 500 mg every 4 hours, notto exceed 6.0 g in 24 hours. For a 200-pound person, these doses areequivalent to about 11 mg/kg, 5.5 mg/kg, and 2.75 mg/kg, respectively.Veterinary dosages are based on a g/kg weight ratio similar to that forhumans. To minimize adverse effects from a too rapid removal of ironfrom the body, siderophore dosages should be spread out over time, wherethe concentration of siderophore in the body is maintained at a moderatelevel during the course of deferration therapy. A dosage regimen ofabout 15 mg/kg/hr for strong chelators is generally recognized as amaximum. Due to the low toxicity of anguibactin and its iron-bindingbehavior similar to other siderophores, it is expected that a similardose regimen would be used for anguibactin when administered to humans.

To determine the ability to excrete chelated iron, a procedure similarto that generally used to evaluate iron chelators is employed. Traceamounts of ⁵⁹ FeCl₃ are incubated in vitro with about 10 mg anguibactinand subcutaneously injected into normal subjects such as rats.Cumulative excretion of radioiron is measured during the subsequent weekin urine and feces. Weak iron chelators are not able enhance ⁵⁹ Feexcretion relative to a control injected with ⁵⁹ FeCl₃ only. Strongchelators such as anguibactin and deferoxamine enhance excretion ofradioiron. Evaluation of the distribution of radioiron in urine andstool provides an indication of whether the siderophore-bound iron isremoved from plasma by kidneys (urine) or by the liver followed byexcretion to the bile (stool).

To determine whether anguibactin functions to remove iron from the ironpool in the body, radioiron is administered to subjects, followed by anevaluation of the distribution of radioiron in the reticuloendothelialsystem and blood components. Again, anguibactin is administered as anaqueous solution. Appropriate radioprobes include ⁵⁹ Fe-ferritin and ⁵⁹Fe-denatured red blood cells (⁵⁹ Fe-DRBC) for targeting the parenchymaland reticuloendothelial iron stores, respectively.

Clinical conditions in which iron chelator therapy is employed aresimulated by using hypertransfused subjects, such as rodents. Forexample, anguibactin is administered as a constant subcutaneous infusionat a rate of 0.1 mg/kg/hr in rats. Infusion is performed using anosmotic minipump implanted between the scapulae. The rate of fluiddelivery of the osmotic pump is 10 microliters per hour. Infusion isstarted at the time of ⁵⁹ Fe labeling and continued for 24 hours. Urineand stool are collected and the amount of radioiron excreted isdetermined. In separate experiments, anguibactin is administered orallyfollowed by assessment of radioiron excretion in urine and stool.

Controls for both hypertransfused and orally-dosed subjects arenon-dosed subjects as well as subjects receiving another siderophoresuch as deferoxamine at the same dose.

Hypertransfusions are performed by two intravenous infusions of 2 mL ofpacked cells suspended in 1 mL physiological saline per 100 g bodyweight of the subject. Infusions are made through a catheter placed inthe right jugular vein under halothane anesthesia. The catheter canconsist merely of a silastic tip inserted into the vein and welded to alength of polyethylene PE-50 tubing tunneled subcutaneously to exit atthe nape of the neck. The catheters should be implanted at least 72hours before beginning infusion. Infusions can be made using, forexample, a Harvard infusion pump. Infusion of packed cells is performedat four days and again at one day before administration of radiolabelediron.

⁵⁹ Fe-DRBCs are prepared by injecting rats with 100-200 μCi of ⁵⁹Fe-citrate five days prior to harvesting the cells. The rats are theninjected with 50-100 μCi of ⁵⁹ Fe-citrate to maintain specificradioactivity at about 0.05 to 0.1 μCi/mg hemoglobin. Blood is removedin volumes of about 1 mL. After washing the blood cells, the cells areresuspended and subjected to 40° C. for 15 min to heat-denature them.After heat-denaturation, the cells are washed again and suspended insaline buffer at a concentration of 5 mL per mL.

⁵⁹ Fe-ferritin is prepared by injecting 10-200 μCi of ⁵⁹ Fe-citrate intorats which have been given 12 mg of Fe-dextran 7 days before. After 24hours, the rats are sacrificed and ferritin therefrom prepared accordingto the method of Bjorklid and Helgeland, Biochem. Biophys. Acta 221:583(1970).

As stated hereinabove, the anguibactin molecule appears to be stable inpH environments as low as pH 3. Nevertheless, the molecule undergoesacid hydrolysis to at least a partial degree as the environment becomesmore acidic. Since the pH of the stomach lumen is highly acidic, one wayto administer anguibactin orally without the possibility of excessiveacid hydrolysis occuring before the compound is absorbed in the gut isto encapsulate it into microcapsules able to pass through the stomachand dissociate in the small intestine. Similar methods are employed inthe art for administering so-termed "time-release" pharmacologicalagents. For example, anguibactin could be encapsulated using an entericcoating applied around a tablet, a capsule, or individual particless,droplets, or granules. One method involves the use of gelatine capsulescoated with cellulose acetate phthalate/diethylphthalate copolymer,which protects the gelatin from water under acid conditions such asfound in the stomach. Under the neutral to slightly alkaline conditionsfound in the intestines, the coating becomes deprotonated and hencevulnerable to attack by water. Another coating example is polymerichydrogels which are resistant dissociation under acidic conditions.

Immobilized Anguibactin

Anguibactin has a chemical structure that would enable it to beimmobilized on an insoluble substrate and used, for example, in theconstruction of columns through which an iron-containing liquid ispercolated for the purpose of removing iron therefrom. In this manner,for example, fluids could be deferrated outside a patient's body, wherethe fluid is either removed from the body via catheter or the like,passed through said column, then returned to the body, or passed as atherapeutic agent through said column to effect deferration beforeadministration to the patient.

Binding of anguibactin to a substrate would in most cases require abifunctional "spacer" or "linker" compound serving to chemically bondthe siderophore to the sustrate in a manner known in the art ofconjugating chemically active molecules to substrates. The linker shouldbe bound to anguibactin in a location on the siderophore molecule notparticipating in the bonding of Fe³⁺ or in a location that would notalter the configuration of the molecule and render it either incapableor weakly capable of chelating iron. Although linkers should bebifunctional, they need not have the same reactive group on each end.

To bond anguibactin to a substrate, it is important that moleculescomprising the substrate have substituent groups available toparticipate in reactions by which anguibactin is conjugated via thelinker to the substrate. Such groups can include carboxylic acid groups,amides, aldehydes, halogens, hydroxyls, sulfonates, azides, and otherreactive groups known in the art that will react with complementaryreactive groups on the linker molecules. In conjugating the anguibactinto the substrate, a reactive group on one end of the linker reacts withan available reactive group on the substrate; a reactive group on theother end of the linker reacts with the anguibactin in a manner whereinthe siderophore retains its ability to bind iron. Representativereactions known in the art by which the linker becomes bonded to thesubstrate include formation of esters, amides; amino, amidino, or diazolinkages, ethers, sulfonamides, and the like. The linker can include ahydrocarbon or other chain serving to space the anguibactin, whilebonded to the substrate, away from the substrate surface.

Suitable substrates for the above include hydrophilic gels such asagarose, alginate, and polyacrylamide, plastics such as polystyrene andnylon, glass, silica gel, ion exchange resins, carbohydrate polymerssuch as cellulose, dextran, and sephadex. The substrate is preferably ina particulate form or other form amenable to liquid percolationtherethrough. A relatively high surface area is preferred to ensure amaximal number of conjugates.

Once the siderophore is bonded to the substrate, the substrate istypically packed into one or more columns through which the liquid to bedeferrated is passed for the purpose of iron removal. Representativeareas in which such a technology could be used is in deferration ofhemodialysate (wherein iron in dialysate has been known to causeprecipitation problems in dialysis equipment) and of liquidpharmaceuticals. Since many bacteria are dependent on a source of ironfor growth, removal of virtually all the iron from a liquidpharmaceutical or other liquid such as for food or cosmetic use canrender the liquid much less able to support bacterial growth and,therefore, more resistant to certain types of spoilage.

Having described the principles of my invention with reference toseveral preferred embodiments, it should be apparent to those ofordinary skill in the art that the invention may be modified inarrangement and detail without departing from such principles. I claimas my invention all such modifications as come within the true spiritand scope of the following claims.

I claim:
 1. A method for inhibiting the uptake of ferric iron by apopulation of living eukaryotic animal cells that is in contact with anaqueous medium comprising ferric iron, the method comprising:addinganguibactin having substantially no bound ferric iron to the aqueousmedium in an amount sufficient to chelate at least a portion of theferric iron in the aqueous medium; and after adding the anguibactin,incubating the cells contacted by said medium for a time sufficient forthe anguibactin to chelate at least a portion of the ferric iron in theaqueous medium, thereby rendering said ferric iron unavailable foruptake by said cells.
 2. A method for reducing the concentration offerric iron present in a vertebrate animal subject, the methodcomprising:adding anguibactin having substantially no bound ferric ironto an aqueous carrier liquid physiologically acceptable to said animalsubject to form an aqueous pharmacological solution of anguibactin; andadministering at least one dose of said aqueous solution of anguibactinto said animal subject to chelate at least a portion of the ferric ironpresent in said animal subject.
 3. A method as recited in claim 2wherein the anguibactin is added to pyrogen-free sterile water servingas said aqueous carrier liquid.
 4. A method as recited in claim 2wherein the anguibactin is added to pyrogen-free sterile isotonic salinesolution serving as said aqueous carrier liquid.
 5. A method as recitedin claim 2 wherein the anguibactin is administered subcutaneously tosaid animal subject.
 6. A method as recited in claim 2 wherein theanguibactin is administered intramuscularly to said animal subject.
 7. Amethod for reducing the concentration of ferric iron present in the bodyof a vertebrate animal subject, comprising orally administering at leastone pharmacologically effective dose of anguibactin that has beentreated in a manner serving to protect the anguibactin from hydrolysisunder acid conditions and make the anguibactin available for absorptioninto the body of said animal subject under neutral and alkalineconditions.